The nuclear pore complex, a 100-megadalton complex comprised of 100 proteins in multiple copies, is the structure through which the import, export and cycling of a large number of diverse protein and RNA substrates occur. The process is regulated for substrates above~50 kDa. This proposal concerns the structure and function of the vertebrate NPC. The first aim seeks to dissect the molecular interactions and functions of two members of the NPC, Nup98 and Nup153. Both proteins are localized to the nuclear face of the NPC; evidence has linked Nup98 with export and Nup153 with import. The sites of contact with targeting molecules and other members of the NPC will be mapped. The second aim utilizes annulate lamellae, structures that form spontaneously in egg cytoplasm that resemble NPCs but are easier to manipulate. The goal is to tease apart this complex to identify molecular interactions of known NPC proteins and to identify new ones. The formation of the annulate lamellae will also be studied in vitro, which would also provide structural information. In the final aim, the P.I. will utilize a novel assay in which proteins bound to ligands are tagged, the tagged molecules are added to a annulate lamellae constitution reaction, and involved tagged proteins identified. This assay will be used to identify novel NPC proteins, or to test for interactions with known NPC constituents.